Glucose concentration is an important indicator in clinical diagnosis as an important marker for diabates. In addition, quantification of glucose concentration is an important indicator for monitoring the process of fermentation production using bacteria. Conventionally, quantification of glucose has been performed by enzymatic methods using glucose oxidase (GOD) or glucose-6-phosphate dehydrogenase (G6PDH). These days, the use of glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme (PQQGDH) is attracting attention in glucose quantification. PQQGDH has highly oxidative activity towards glucose and it does not require oxygen as an electron acceptor since PQQGDH bears its coenzyme. Thus PQQGDH is a promising enzyme to be applied on glucose assays, for example, as a recognition devise of a glucose sensor.
PQQGDH is a glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme, and catalyzes the reaction of oxidizing glucose to produce gluconolactone. Two types of PQQGDHs are known: membrane-bound and water-soluble. Membrane-bound PQQGDH is a single-peptide protein with an approximate molecular weight of 87 kDa, and is found in a wide variety of gram-negative bacteria. On the other hand, water-soluble PQQGDH has been found in some strains of Acinetobacter calcoaceticus (Biosci. Biotech. Biochem. (1995), 59 (8), 1548–1555), and its structural gene has been cloned and its amino acid sequence determined (Mol. Gen. Genet. (1989), 217:430–436). Water-soluble PQQGDH derived from A. calcoaceticus is a water-soluble homodimer enzyme consisting of two 50 kDa subunits. It requires PQQ and Ca2+ for its activity and shows the enzyme activity of as high as 2200 U/mg–7400 U/mg. Isoelectric points are approximately 9.2 and 10.2 for apoenzyme without bound PQQ and holoenzyme, respectively, indicating that the enzyme is a basic protein (K. Matsushita, et al. (1995) Biosci. Biotech. Biochem., 59, 1548–1555). In addition, the results of X-ray structural analysis of water-soluble PQQGDH have been published and reveal the conformation of water-soluble PQQGDH and estimated location of PQQ and Ca2+ (A. Oubrie, et al. (1999) J. Mol. Bio., 289, 319–333, A. Oubrie, et al. (1999) The EMBO Journal, 18 (19), 5187–5194, and A. Oubrie, et al. (1999), PNAS 96 (21), 11787–11791).
The wild type water-soluble PQQGDH shows a marked reduction in its activity by substrate inhibition under the glucose concentration of 100 mM or more. For this reason, quantitative measurement of the substrate concentration is difficult under high substrate concentration. The mechanism of substrate inhibition is not yet known.
Hence, the present invention is aimed at providing a modified water-soluble PQQGDH which shows a little reduction of enzyme activity due to substrate inhibition.